Mass spectrometer is an analytical device for measuring a mass of a compound. It generally determines molecular weight of a compound by measuring the value of mass-to-charge (m/z) by ionizing the compound by charging. There are many methods to ionize a compound such as electron ionization using electron beams, high speed collision of atoms, a method using laser, and a method to spray a specimen into an electric field.
For a biochemical substance with an extremely large mass such as proteins and nucleic acids, MALDI-T of Mass Spectroscopy (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectroscopy; hereinafter MALDI), which uses laser, has been used and various analytic devices of this kind have been recently developed and are commercially available at present. These devices can measure molecular weight of polymers with molecular weight of 300 kDa or higher by using matrix which not only assists the transfer of energy to a substance to be analyzed but also facilitates ionization of the substance. Besides, analyses of specimens at the level of femto moles are also possible due to their relatively high sensitivity, and also help to greatly reduce the breakage of a compound during ionization process thereby enabling the analyses of mixtures.
For the MALDI-assisted mass spectrometry, specimens are prepared as follows.
(1) A small amount of matrix solution is added onto a target board made of a metal plate, dried and then a specimen solution to be analyzed is further added on top of it and dried; or
A matrix substance is mixed with a specimen solution, placed onto a target plate and then crystallized.
(2) The area of the matrix and crystallized specimen is irradiated with laser and the specimen becomes desorbed/ionized being assisted with the matrix.
Typical mass spectrometer has a structure that it applies an electric field between the target board where a specimen is located and a sensor for mass analysis so that ionized specimen can be moved into a sensor due to the difference in potentials. In case that the electric charge of a specimen is already known the mass of the specimen can be analyzed based on variables such as the time required for the specimen to reach the sensor.
The above MALDI method is very useful for mass analysis but it is still necessary to select a matrix substance suitable for ionization depending on the properties of a specimen. Examples of the matrix substance are nicotinic acid, cinnamic acid, 2,5-dihydroxybenzoic acid and the like. These matrix substances are known to generate protons by absorbing energy being irradiated and bind them to a specimen thereby facilitating the ionization of the specimen. However, the exact mechanism of the ionization is yet to be elucidated and therefore it is challenging to select a suitable matrix substance for a given specimen.
Besides, MALDI has a disadvantage that its use is largely limited to substances having a molecular weight greater than 1,000 Da because low molecular weighted matrix substance and matrix decomposed product are indicated on the mass spectrometry spectrum in the course of ionization of a specimen by a laser-activated matrix.
In addition, the ionization of a decomposed product is determined according to the selection of a matrix and thus it becomes necessary to select a most suitable matrix substance and it becomes difficult to perform an analysis for an unknown substance in a mixture (G. Suizdak, 1. Ion sources and sample introduction, In: Mass spectroscopy for biotechnology, Academic press, 1996, p 13).
Further, MALDI method has another disadvantage that the spatial distribution of a crystallized specimen obtained via specimen preparation process is not uniform and therefore the amount of a specimen being excited by laser varies depending on the location of irradiation. Therefore, for appropriate quantitative analysis, the various results obtained by irradiating various locations are interpreted statistically. A widely known method for quantitative analysis via MALDI method is to measure spectrum of a compound having an almost identical structure as the one to be analyzed by incorporating an internal standard substance labeled with a radioisotope in a predetermined ratio (M. J. Kang, E. Heinzle, Rapid Communications in Mass Spectrometry, 15 (2001) 1327-1333). However, even with the above method, it is still difficult to perform an accurate quantitative analysis of a specimen via MALDI method.
As a mass analysis method using laser as an energy source for desorption/ionization of a specimen without using matrix there is introduced DIOS MS (Desorption Ionization on Silicon Mass Spectroscopy; hereinafter DIOS). In general, DIOS is a method for analyzing mass of a specimen using porous silicon as a target without matrix. The porous silicon is manufactured by electric etching and DIOS becomes possible by adjusting porosity and degree of oxidation. The porous silicon used in DIOS is expected to provide ionization of a specimen by absorbing laser energy as in the case of matrix but the exact energy transfer pathway for desoprtion/ionization of a specimen is not known yet.
It is known that DIOS enables to perform a quantitative analysis, without using matrix, of substances with high molecular weight such as proteins and nucleic acids as well as compounds with relatively low molecular weight (J. Wel, J. M. Burlak, G. Suizdak, Nature, 399 (1999) 243-246; W. G. Lewis, Z. Shen, M. G. Finn, G. Suizdak, International Journal of Mass spectrometry, 226 (2003) 107-116; U.S. Pat. No. 6,288,390 B1).
Meanwhile, in case of DIOS, a specimen solution penetrates the porous structure of a board and becomes crystallized in the course of injecting a specimen solution on the porous silicon board thus making it difficult to limit the size of crystals and also hard to make the crystal center of a specimen correspond to a point of laser irradiation. For this reason, it is almost impossible to perform a quantitative analysis through 1 to 2 times of measurements of a specimen. Since DIOS is only applicable to silicon, it cannot select an energy transfer medium suitable for various kinds of specimens. Further, an effective desorption/ionization becomes difficult because the laser energy can be transferred via a two-dimensional way.
Therefore, to resolve the above-mentioned problems, the present invention provides a method for a nanowire-assisted laser desorption/ionization mass spectrometric analysis wherein the nanowire, which is used instead of the traditional porous silicon, can fix a specimen and enables to perform mass analysis of a specimen without using a matrix solution while effectively transferring laser energy to a specimen to be irradiated.